PROJECT SUMMARY/ABSTRACT Altered regulation of lipid and glucose homeostasis, most often in the setting of insulin resistance and obesity, is central to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Because current management options remain limited, the discovery of new metabolic pathways will serve to identify novel opportunities for pharmacologic intervention. This research proposal addresses the unanswered question of whether membrane phospholipids regulate nutrient homeostasis. Our long-term goal is to understand how phospholipid-mediated metabolic control can be leveraged for therapeutic purposes. The objective of this research is to determine the molecular mechanisms whereby sensing of membrane phosphatidylcholines by phosphatidylcholine transfer protein (PC-TP) is translated into metabolic control by thioesterase superfamily member 2 (Them2), a mitochondria-associated long chain acyl-CoA thioesterase. The central hypothesis is that key regulatory events occur in the skeletal muscle when PC-TP binds specific membrane phosphatidylcholine molecular species and then activates Them2. The rationale is that regulation of skeletal muscle metabolism by Them2 should yield new insights into hepatic insulin resistance and steatosis. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To demonstrate that Them2 controls lipid and glucose metabolism in skeletal muscle; 2) To define mechanisms whereby Them2 in skeletal muscle promotes hepatic steatosis; and 3) To elucidate the molecular determinants of Them2 activity and regulation by PC-TP. In Aim 1, mouse models will be used to determine mechanisms whereby Them2 regulates fatty acid and glucose metabolism in skeletal muscle and promotes insulin resistance in response to overnutrition. Cell autonomous functions of Them2 will be gleaned from systematic studies in cultured myotubes. Aim 2 will establish the mechanisms in high fat fed mice whereby Them2 in skeletal muscle promotes hepatic insulin resistance and steatosis. Cultured hepatocytes will be used to determine whether Them2-dependent myokines or extracellular vesicles released from myotubes control hepatic lipid and glucose metabolism. Aim 3 will determine structural characteristics that enable Them2 to respond to changes in membrane phosphatidylcholine composition. Small molecules designed to bind and inhibit Them2 will be used as probes to characterize Them2 function and interactions with PC-TP using an array of biophysical techniques. Phosphatidylcholine-dependent conformational changes in PC-TP will be leveraged to identify specific motifs that are critical for Them2 activity and stability. The interacting domains of Them2 and PC-TP will be identified and confirmed by mutational analyses. Overall, this proposal will elucidate new mechanisms of phospholipid-mediated metabolic regulation that control hepatic nutrient metabolism, which is significant because the fatty acyl composition of membrane phosphatidylcholines varies in health and disease. These studies are expected to establish Them2 as a tractable target for the management of NAFLD.